Analysis of novel polar auxin transport pathway components in Arabidopsis

拟南芥中新型极性生长素转运途径成分分析

• 批准号: 2214387
• 金额: --
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2214387
• 关键词: Analysis; novel; polar; auxin; transport

The plant hormone auxin plays a key role in coordinating diverse aspects of plant development, contributing to crop productivity. This includes the development of the shoot, root, flowers, seeds, fruit, stem elongation, tissue differentiation. An important aspect of the mode of action of auxin is its polar transport within developing tissues, to establish concentration gradients that create positional information to allow spatial regulation of gene expression patterns. The most important components of the directional auxin transport mechanism are the familiy auxin efflux carriers (the PIN proteins), some of which localize to specific faces of the plasmamembrane, to control directional auxin efflux. Rapid changes in cell polarity involve clathrin-mediated endocytosis and recycling of PINs. Auxin itself inhibits this recycling, resulting in an accumulation of PIN proteins at the plasmamembrane, so promoting its own efflux. While the endocytic model accounts for the dynamic relocalization of PINs to different surfaces of the cell, it does not explain mechanistically how PIN proteins are delivered to the plasmamembrane following their translation in the endoplasmic reticulum (ER). We have identified a novel mutant defective in the polar auxin transport pathway. The gene involved is VAMP714, identified as both gain of function and loss of function mutants (Lindsey lab, unpublished). Bioinformatics analysis suggests the gene is an R-SNARE, and evidence suggests it is localised to the trans-Golgi vesicles, and co-localises with PIN proteins. vamp714 mutants fail to correctly localise PIN proteins to the plasmamembrane, and show reduced polar auxin transport (Lindsey lab, unpublished). We therefore hypothesise that VAMP714 is essential for the delivery of PIN proteins to the plasmamembrane. The aim of this project is to characterise the essential role of the Arabidopsis thaliana VAMP714 and related VAMP proteins in delivering PIN proteins to the plasmamembrane. Specific objectives are: 1) to characterise the localisation of VAMP7 family proteins in Arabidopsis cells, by co-localization with subcellular compartment markers; 2) to determine whether VAMP7 family protein localisation forms part of the endocytic recycling pathway, using recycling inhibitors such as latrunculin B and Brefeldin A; 3) to characterise in detail the meristem phenotype of vamp7 family mutants. Preliminary data suggest that VAMP714 is required for maintenance of the stem cell niche in the root meristem; 4) to characterise PIN localization in vamp7 family mutants; 5) to characterise changes in gene transcription in vamp7 family mutants with a focus on the analysis of key genes known to be a) auxin-regulated in the meristem and b) regulators of meristem identity and function. The project provides training in New Ways of Working, and specifically in advanced bioimaging, bioinformatics and transcriptomics. There is also the opportunity to use information from the project to develop further the predictive mathematical model we have developed and published that allows us to understand the crosstalk between auxin, its synthesis and transport (via PIN proteins), and other genes and signalling components in the root meristem (e.g. Moore et al. 2015 New Phytologist 207(4): 1110-1122).

植物激素生长素在协调植物发育的各个方面发挥着关键作用，有助于提高作物生产力。这包括芽、根、花、种子、果实、茎伸长、组织分化的发育。生长素作用模式的一个重要方面是其在发育组织内的极性运输，以建立浓度梯度，从而产生位置信息，从而允许基因表达模式的空间调节。定向生长素转运机制最重要的组成部分是家族生长素流出载体（PIN 蛋白），其中一些定位于质膜的特定面，以控制定向生长素流出。细胞极性的快速变化涉及网格蛋白介导的内吞作用和 PIN 的回收。生长素本身会抑制这种循环，导致 PIN 蛋白在质膜上积累，从而促进其自身的外流。虽然内吞模型解释了 PIN 动态重新定位到细胞不同表面的情况，但它并没有从机制上解释 PIN 蛋白在内质网 (ER) 中翻译后如何被传递到质膜。我们发现了一种在极性生长素运输途径中存在缺陷的新型突变体。所涉及的基因是 VAMP714，被鉴定为功能获得和功能丧失突变体（Lindsey 实验室，未发表）。生物信息学分析表明该基因是一个 R-SNARE，并且有证据表明它定位于跨高尔基体囊泡，并与 PIN 蛋白共定位。 vamp714 突变体无法将 PIN 蛋白正确定位到质膜，并显示极性生长素转运减少（Lindsey 实验室，未发表）。因此，我们假设 VAMP714 对于将 PIN 蛋白递送至质膜至关重要。该项目的目的是表征拟南芥 VAMP714 和相关 VAMP 蛋白在将 PIN 蛋白递送至质膜中的重要作用。具体目标是：1) 通过与亚细胞区室标记的共定位来表征 VAMP7 家族蛋白在拟南芥细胞中的定位； 2) 使用诸如latrunculin B和Brefeldin A等回收抑制剂来确定VAMP7家族蛋白定位是否构成内吞回收途径的一部分； 3）详细表征vamp7家族突变体的分生组织表型。初步数据表明，VAMP714 是维持根分生组织干细胞生态位所必需的； 4) 表征 vamp7 家族突变体中的 PIN 定位； 5) 表征 vamp7 家族突变体中基因转录的变化，重点分析已知的关键基因：a) 分生组织中的生长素调节因子和 b) 分生组织特性和功能的调节因子。该项目提供新工作方式的培训，特别是高级生物成像、生物信息学和转录组学方面的培训。还有机会利用该项目的信息进一步开发我们已经开发和发布的预测数学模型，使我们能够了解生长素、其合成和运输（通过 PIN 蛋白）以及根分生组织中的其他基因和信号成分之间的串扰（例如 Moore 等人 2015 New Phytographer 207(4): 1110-1122）。